Electrical stimulation is a potential therapy for gastric disorders. Here we describe our surgical procedure for the implantation of patch electrodes on the muscle wall of the stomach of both male and female rats for use in chronic studies looking at the long-term effects of stimulation on physiology and behavior. The surgery is well tolerated by the subjects as rats return to pre-surgical body weight and normal solid chow intake within 7 to 10 days post-surgery. In our hands, rats implanted using the methods described below have been continuously run in feeding and motility experiments for 8 to 12 weeks with minimal to no loss of subjects. When failure does occur, it is typically a result of back mount failure at the site of attachment of the overhead tether to the back mount.

Gastric electrical stimulation (GES) is used clinically to promote proximal GI emptying and motility. In acute experiments, we measured duodenal motor responses elicited by GES applied at 141 randomly chosen electrode sites on the stomach serosal surface. Overnight-fasted (H 2 O available) anesthetized male rats ( n = 81) received intermittent biphasic GES for 5 min (20-s-on/40-s-off cycles; I = 0.3 mA; pw = 0.2 ms; 10 Hz). A strain gauge on the serosal surface of the proximal duodenum of each animal was used to evaluate baseline motor activity and the effect of GES. Using ratios of time blocks compared with a 15-min prestimulation baseline, we evaluated the effects of the 5-min stimulation on concurrent activity, on the 10 min immediately after the stimulation, and on the 15-min period beginning with the onset of stimulation. We mapped the magnitude of the duodenal response (three different motility indices) elicited from the 141 stomach sites. Post hoc electrode site maps associated with duodenal responses suggested three zones similar to the classic regions of forestomach, corpus, and antrum. Maximal excitatory duodenal motor responses were elicited from forestomach sites, whereas inhibitory responses occurred with stimulation of the corpus. Moderate excitatory duodenal responses occurred with stimulation of the antrum. Complex, weak inhibitory/excitatory responses were produced by stimulation at boundaries between stomach regions. Patterns of GES efficacies coincided with distributions of previously mapped vagal afferents, suggesting that excitation of the duodenum is strongest when GES electrodes are situated over stomach concentrations of vagal intramuscular arrays, putative stretch receptors in the muscle wall.

ObjectiveIn spite of its apparent promise, bioelectric modulation of the vagal innervation of the stomach has had limited success modifying ingestive behavior. But, importantly, stimulation parameters in previous bioelectrical applications were arbitrarily employed without information as to their stimulus‐response relationships or optimal locations. With this in mind, we established a model of chronic vagal nerve stimulation (VNS) where we record GI motility continuously in unanesthetized, freely moving rats while their intake is monitored as they consume chow and water ad libitum (automated food and liquid intake monitors; BioDAQ, Research Diets Inc.).MethodsA patch electrode (MicroProbes) was attached to the muscle wall of the proximal forestomach of healthy adult male Sprague‐Dawley rats at a site known to contain the highest density of vagal mechanoreceptors. A strain gauge was similarly attached to the muscle wall of the antrum. Rats were maintained in their home cages equipped with BioDAQ feeders, while continuously tethered to overhead commutators. Following recovery, the feeding and drinking behavior as well as antral motility of individual rats was continuously monitored and recorded with and without concomitant VNS (pulse parameters, derived from acute studies: 0.3mA/0.2ms/10Hz; 20s On:40s Off). Subjects were exposed to each of the following four conditions on different days at the onset of lights off: a 2 h fast, a 2 h fast while being stimulated, 2 h ad lib access to chow, and 2 h ad lib access to chow while being stimulated. In a separate study, 2 days of strain gauge activity was continuously recorded: 24 h without stimulation followed by 24 h with stimulation.ResultsRats recovered quickly following surgery. Compared to the 2 h fasted condition, there was a 17% increase in antral peristalsis (change in amplitude and frequency expressed as area under the curve) when subjects were fasted while being stimulated; similarly, there was an 18% increase in antral peristalsis when subjects were stimulated while consuming chow compared to 2 h intake of chow without stimulation. The same pattern was observed in the 2 d study with a 23% increase in antral activity during 24 h of stimulation compared to 24 h pre‐stimulation. Interestingly, a 12% increase in total chow intake occurred when stimulated for 24 h while no difference was noted in water intake between the two conditions.ConclusionsInitial exposure to VNS results in a modest increase in antral activity with and without the presence of food in the stomach. Analyses are underway to tease apart the effect of stimulation on motility in relation to meal size. Finally, these findings confirm that our chronic VNS model has considerable potential for determining optimal stimulation parameters necessary for making meaningful changes in gut motility.Support or Funding InformationSPARC/NIH Office of the Director OT2 OD023847NIDDK/NIH R01 DK027627

PurposeThe gut communicates with the brain, allowing the gastrointestinal state to influence cognition and emotion and vice versa. In the resting state, gastric electrical activity has been shown to be synchronized with the blood‐oxygen‐level‐dependent (BOLD) signal in the so‐called gastric network in humans [1]. However, the finding has been rarely replicated. Here, we explored the gut‐brain synchrony in rats. Whole‐brain functional magnetic resonance imaging (fMRI) was acquired simultaneously with electrogastrogram (EGG) recording. Cross‐correlation between EGG and BOLD signals was used to map resting state networks influenced by gastric activity.MethodBrain fMRI was performed on three SD rats together with multi‐channel EGG recording. Each rat was trained to consume diet gel enriched with Gadolinium contrast media. Before the experiment, the rat was fed with 5g diet gel. After the feeding, the animal was anesthetized with continuous dexmedetomidine and isoflurane. The multi‐channel EGG signal was recorded using an electrophysiological recording system (Tucker Davis Technologies Inc.) together with fMRI scans using a 7‐tesla small‐animal MRI system (BioSpec 70/30, Bruker). The principal component analysis was applied to EGG signals. The correlation between the voxel‐wise fMRI signal and the component‐wise EGG signal was calculated, yielding multiple gut‐associated brain networks. The voxel‐wise fMRI signal was also modeled as a linear combination of EGG component time series with varying time shifts.ResultsFMRI was acquired simultaneously with the EGG recording ( Fig. 1A). EGG was denoised and cross‐correlated with the BOLD activity in every voxel to map an EGG‐correlated network (or the gastric network) ( Fig. 1C). The BOLD signal was averaged across voxels in the gastric network. The resulting network‐wide signal was found to be phase‐locked to EGG with a time delay ( Fig. 1B). Accounting for the delay variable across voxels, we mapped an even broader network phase‐locked with EGG ( Fig. 2A). Different EGG‐coupled regions were found to lag behind EGG by different times, ranging from 4 to 12 seconds ( Fig. 2B & C). From the EGG‐coupled BOLD response, the visual and auditory cortex had a relatively longer delay, whereas the somatosensory and anterior cingulate cortex had a shorter delay. The cingulate cortex showed a complex response pattern, likely implying multiple functional roles with respect to the gut.ConclusionIn rats, brain activity is intrinsically synchronized with gastric activity at a specific resting state network. In this network, different brain regions are time‐locked to gastric activity with varying time delays. Speculatively, the pattern of time delay may reflect how gastric information propagates through a cascade of brain regions for sequential processing.Support or Funding InformationThis study was funded by National Institutes of Health’s SPARC ‐ Stimulating Peripheral Activity to Relieve Conditions ‐ program (OT2OD023847).Simultaneous EGG and brain fMRI recording. (A) shows the layout of the 32‐channel electrode array for EGG recording. (B) are examples of EGG and fMRI time series. (C) shows an example of the EGG correlation map. The correlation between EGG and fMRI time series is color‐coded.Figure 1The EGG network considering the phase delays between EGG and fMRI signal. (A) is a summary of the EGG network. The color represents the F statistic of the multiple linear regression. (B) selects six voxels in the EGG network. The curve shows the relation between the F statistic and EGG‐fMRI delays for the selected voxels, which are marked with blue box in (A). (C) is the phase delay map that delineates the phase differences between EGG and fMRI time series.Figure 2

BACKGROUNDThe discovery that the stomach secretes the hormone leptin, plus the finding that vagal afferents express receptors for leptin, in addition to the recognized role of adipocytes in the secretion of the adipokine, have initiated extensive investigations into leptin’s possible roles in the control of feeding. The gastric secretion of leptin and its binding sites on vagal afferents suggest the possibility that gastric electrical stimulation (GES) might produce leptin secretion and modulate visceral sensory information arising from the stomach and relayed to the brain. Such a pathway might have therapeutic potential for treating GI disorders (gastroparesis, eating disorders, etc.).AIMSTo explore or map defined gastric regional patterns of leptin release, secretion of the hormone to GES at different sites was measured in terms of both amplitude and time course.METHODSIn fasted (18 hrs.), anesthetized (Isoflurane) SD rats (n = 49), patch electrodes were sutured on ventral stomach wall, a strain gauge was attached to duodenum, and a catheter was inserted into left femoral artery. Stimulation (biphasic, 0.3mA, 0.2ms, 10Hz, 20s‐on‐40s‐off; 5 cycles) was applied from 0 to 5 min. The gastric antrum, corpus and forestomach were each divided into three regions corresponding to the distance between the lower esophageal sphincter (LES) and the greater curvature (GC); each of the three gastric compartments (verified post mortem) was stimulated near the LES, at a mid‐point, and near the GC. Multiple blood samples (0.15ml/each) were collected to measure leptin concentrations.RESULTSStimulation of much of the antrum (mid‐and near‐GC antral regions) evoked fast, robust and long‐lasting leptin secretion. Stimulation of the corpus in a more limited area (mid‐corpus) produced moderate, long‐lasting leptin secretion. Stimulation of a limited forestomach area (mid‐forestomach) yielded a mild, short‐interval leptin secretion. For the effective loci, leptin secretion amplitude changes in post‐stim time points of 5, 15, and 30 min, respectively, compared to control values, were: Mid‐antrum: +58%, +70%, +48%; Near‐GC antrum: +43%, +70%, +33%; Mid‐corpus: +25%. +38%, +38%; and Mid‐forestomach: +38%, +22%, +8%.DISCUSSIONGastric leptin is secreted by both exocrine and endocrine pathways. Antrum sensitivity to stimulation may reflect higher density of leptin‐secretory epithelium, corpus may have moderate density leptin‐related epithelium. Without leptin‐related epithelium in forestomach, leptin release may be caused indirectly.CONCLUSIONOur findings reveal that GES in different stomach regions can produce strikingly different leptin secretion patterns. Selectively modulating leptin secretion with GES applied to different stomach regions could be used as a neuromodulation strategy to treat GI disorders such as gastroparesis, obesity, and various eating disorders.Support or Funding InformationFUNDING: SPARC/NIH OT2 OD023847NIDDK/NIH R01 DK027627

Vagus nerve stimulation (VNS) is an emerging bioelectronic therapy for regulating food intake and controlling gastric motility. However, the effects of different VNS parameters and polarity on postprandial gastric motility remain incompletely characterized.

We sought to determine whether design of carbohydrate-based microspheres to have different digestion rates, while retaining the same material properties, could modulate gastric emptying through the ileal brake. Microspheres made to have three slow digestion rates and a rapidly digested starch analogue (maltodextrin) were administrated to rats by gavage and starch contents in the stomach, proximal and distal small intestine, and caecum were measured 2 h post-gavage. A stepwise increase in the amount of starch retained in the stomach was found for microspheres with incrementally slower rates of digestion. Postprandial glycaemic and insulinaemic responses were incrementally lower for the different microspheres than for the rapidly digestible control. A second-meal effect was observed for slowly digestible starch (SDS) microspheres compared to glucose. Thus, dietary slowly digestible carbohydrates were designed to elicit incremental significant changes in gastric emptying, glycaemic and insulinaemic responses, and they may be a means to trigger the ileal brake.

This protocol describes the methods used to trace and enable morphometric quantification of vagal afferent neurites in the rat stomach. A mixture of dextran conjugates was injected into the nodose ganglia of young adult Sprague-Dawley rats and after a survival period of 14 days for optimal tracer transport, stomachs were removed and processed as whole mounts. ABC-DAB was used to create a permanent gold-brown stain of all labeled afferent neurites. Subgroups of samples were also counterstained with either the panneuronal chromogen cuprolinic blue or with nNOS antibodies and steel gray chromogen to label nitrergic cells.

BEFORE STARTING A simple approach to identify the influence of left vagal stimulus pulse parameters on vagal and gastric electrical activity in rat Matthew Ward , Thomas V Nowak , Zhenjun Tan , Bartek Rajwa , Robert Phillips , Terry L Powley Weldon School of Biomedical Engineering (Purdue University, West Lafayette, IN) and Indiana University School of Medicine (Indianapolis, IN), Indiana University School of Medicine (Indianapolis, IN), Dept. of Psychological Sciences (Purdue University, West Lafayette, IN), Bindley Bioscience Center (Purdue University

Brain−gut neural communications have long been considered limited because of conspicuous numerical mismatches. The vagus, the parasympathetic nerve connecting brain and gut, contains thousands of axons, whereas the gastrointestinal (GI) tract contains millions of intrinsic neurons in local plexuses. The numerical paradox was initially recognized in terms of efferent projections, but the number of afferents, which comprise the majority (≈ 80%) of neurites in the vagus, is also relatively small. The present survey of recent morphological observations suggests that vagal terminals, and more generally autonomic and visceral afferent arbors in the stomach as well as throughout the gut, elaborate arbors that are extensive, regionally specialized, polymorphic, polytopic, and polymodal, commonly with multiplicities of receptors and binding sites—smart terminals. The morphological specializations and dynamic tuning of one‐to‐many efferent projections and many‐to‐one convergences of contacts onto afferents create a complex architecture capable of extensive peripheral integration in the brain−gut connectome and offset many of the disparities between axon and target numbers. Appreciating this complex architecture can help in the design of therapies for GI disorders.

Functional magnetic resonance imaging (fMRI) is commonly thought to be too slow to capture any neural dynamics faster than 0.1 Hz. However, recent findings demonstrate the feasibility of detecting fMRI activity at higher frequencies beyond 0.2 Hz. The origin, reliability, and generalizability of fast fMRI responses are still under debate and await confirmation through animal experiments with fMRI and invasive electrophysiology. Here, we acquired single-echo and multi-echo fMRI, as well as local field potentials, from anesthetized rat brains given gastric electrical stimulation modulated at 0.2, 0.4 and 0.8 Hz. Such gastric stimuli could drive widespread fMRI responses at corresponding frequencies from the somatosensory and cingulate cortices. Such fast fMRI responses were linearly dependent on echo times and thus indicative of blood oxygenation level dependent nature (BOLD). Local field potentials recorded during the same gastric stimuli revealed transient and phase-locked broadband neural responses, preceding the fMRI responses by as short as 0.5 s. Taken together, these results suggest that gastric stimulation can drive widespread and rapid fMRI responses of BOLD and neural origin, lending support to the feasibility of using fMRI to detect rapid changes in neural activity up to 0.8 Hz under visceral stimulation.

BackgroundVagus nerve stimulation (VNS) is an emerging electroceutical therapy for remedying gastric disorders that are poorly managed by pharmacological treatments and/or dietary changes. Such therapy seems promising as the vagovagal neurocircuitry modulates the enteric nervous system to influence gastric functions. MethodsHere, the modulatory effects of left cervical VNS on gastric emptying in rats were quantified using a (i) feeding protocol in which the animal voluntarily consumed a postfast, gadolinium-labeled meal and (ii) a non-invasive imaging method to measure antral motility, pyloric activity and gastric emptying based on contrast-enhanced magnetic resonance imaging (MRI) and computer-assisted image processing pipelines. Key ResultsVagus nerve stimulation significantly accelerated gastric emptying (sham vs VNS: 29.1%1.5% vs 40.7%+/- 3.9% of meal emptied per 4hours), caused a greater relaxation of the pyloric sphincter (sham vs VNS: 1.5 +/- 0.1 vs 2.6 +/- 0.4mm(2) cross-sectional area of lumen), and increased antral contraction amplitude (sham vs VNS: 23.3%+/- 3.0% vs 32.5%+/- 3.0% occlusion), peristaltic velocity (sham vs VNS: 0.50 +/- 0.02 vs 0.67 +/- 0.03mms(-1)), but not its contraction frequency (sham vs VNS: 6.1 +/- 0.2 vs 6.4 +/- 0.2 contractions per minute, P=.22). The degree to which VNS relaxed the pylorus was positively correlated with gastric emptying rate (r=.5887, P<.001). Conclusions & InferencesThe MRI protocol employed in this study is expected to enable advanced preclinical studies to understand stomach pathophysiology and its therapeutics. Results from this study suggest an electroceutical treatment approach for gastric emptying disorders using cervical VNS to control the degree of pyloric sphincter relaxation.

SCOPE Slowly digestible starch (SDS), as a functional carbohydrate providing a slow and sustained glucose release, may be able to modulate food intake through activation of the gut-brain axis. METHODS AND RESULTS Diet-induced obese rats were used to test the effect on feeding behavior of high-fat (HF) diets containing an SDS, fabricated to digest into the ileum, as compared to rapidly digestible starch (RDS). Ingestion of the HF-SDS diet over an 11-week period reduced daily food intake, through smaller meal size, to the same level as a lean body control group, while the group consuming the HF-RDS diet remained at a high food intake. Expression levels (mRNA) of the hypothalamic orexigenic neuropeptide Y (NPY) and Agouti-related peptide (AgRP) were significantly reduced, and the anorexigenic corticotropin-releasing hormone (CRH) was increased, in the HF-SDS fed group compared to the HF-RDS group, and to the level of the lean control group. CONCLUSION SDS with digestion into the ileum reduced daily food intake and paralleled suppressed expression of appetite-stimulating neuropeptide genes associated with the gut-brain axis. This novel finding suggests further exploration involving a clinical study and potential development of SDS-based functional foods as an approach to obesity control.

Detection of α-synuclein lesions in peripheral tissues is a feature of human synucleinopathies of likely pathogenetic relevance and bearing important clinical implications. Experiments were carried out to elucidate the relationship between α-synuclein accumulation in the brain and in peripheral organs, and to identify potential pathways involved in long-distance protein transfer. Results of this in vivo study revealed a route-specific transmission of α-synuclein from the rat brain to the stomach. Following targeted midbrain overexpression of human α-synuclein, the exogenous protein was capable of reaching the gastric wall where it was accumulated into preganglionic vagal terminals. This brain-to-stomach connection likely involved intra- and inter-neuronal transfer of non-fibrillar α-synuclein that first reached the medulla oblongata, then gained access into cholinergic neurons of the dorsal motor nucleus of the vagus nerve and finally traveled via efferent fibers of these neurons contained within the vagus nerve. Data also showed a particular propensity of vagal motor neurons and efferents to accrue α-synuclein and deliver it to peripheral tissues; indeed, following its midbrain overexpression, human α-synuclein was detected within gastric nerve endings of visceromotor but not viscerosensory vagal projections. Thus, the dorsal motor nucleus of the vagus nerve represents a key relay center for central-to-peripheral α-synuclein transmission, and efferent vagal fibers may act as unique conduits for protein transfer. The presence of α-synuclein in peripheral tissues could reflect, at least in some synucleinopathy patients, an ongoing pathological process that originates within the brain and, from there, reaches distant organs innervated by motor vagal projections.

ABSTRACTThe fundamental roles that the stomach plays in ingestion and digestion notwithstanding, little morphological information is available on vagal intramuscular arrays (IMAs), the afferents that innervate gastric smooth muscle. To characterize IMAs better, rats were given injections of dextran biotin in the nodose ganglia, and, after tracer transport, stomach whole mounts were collected. Specimens were processed for avidin–biotin permanent labeling, and subsets of the whole mounts were immunohistochemically processed for c‐Kit or stained with cuprolinic blue. IMAs (n = 184) were digitized for morphometry and mapping. Throughout the gastric muscle wall, IMAs possessed common phenotypic features. Each IMA was generated by a parent neurite arborizing extensively, forming an array of multiple (mean = 212) branches averaging 193 µm in length. These branches paralleled, and coursed in apposition with, bundles of muscle fibers and interstitial cells of Cajal. Individual arrays averaged 4.3 mm in length and innervated volumes of muscle sheet, presumptive receptive fields, averaging 0.1 mm3. Evaluated by region and by muscle sheet, IMAs displayed architectural adaptations to the different loci. A subset (32%) of circular muscle IMAs issued specialized polymorphic collaterals to myenteric ganglia, and a subset (41%) of antral longitudinal muscle IMAs formed specialized net endings associated with the serosal boundary. IMAs were concentrated in regional patterns that correlated with the unique biomechanical adaptations of the stomach, specifically proximal stomach reservoir functions and antral emptying operations. Overall, the structural adaptations and distributions of the IMAs were consonant with the hypothesized stretch receptor roles of the afferents. J. Comp. Neurol. 524:713–737, 2016. © 2015 Wiley Periodicals, Inc.

A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days postinjection, animals were euthanized and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran‐labeled axons (135 of 136 sampled) were tyrosine hydroxylase‐positive. Complete postganglionic arbors ( n = 154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/tertiary plexus. Very few CSMG neurons projected exclusively (i.e., ∼100% of an arbor's varicose branches) to myenteric plexus (∼2%) or smooth muscle (∼14%). With less stringent inclusion criteria (i.e., ≥85% of an axon's varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (∼13%) or smooth muscle (∼27%). The majority (i.e., ∼60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that coinnervated extensively (>15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ∼87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively. J. Comp. Neurol. 524:2577–2603, 2016. © 2016 Wiley Periodicals, Inc.

The pylorus is innervated by vagal mechanoreceptors that project to gastrointestinal smooth muscle, but the distributions and specializations of vagal endings in the sphincter have not been fully characterized. To evaluate their organization, the neural tracer dextran biotin was injected into the nodose ganglia of rats. Following tracer transport, animals were perfused, and their pylori and antra were prepared as whole mounts. Specimens were processed to permanently label the tracer, and subsets were counterstained with Cuprolinic blue or immunostained for c-Kit. Intramuscular arrays (IMAs) in the circular muscle comprised the principal vagal afferent innervation of the sphincter. These pyloric ring IMAs were densely distributed and evidenced a variety of structural specializations. Morphometric comparisons between the arbors innervating the pylorus and a corresponding sample of IMAs in the adjacent antral circular muscle highlighted that sphincter IMAs branched profusely, forming more than twice as many branches as did antral IMAs (means of 405 vs. 165, respectively), and condensed their numerous neurites into compact receptive fields (∼48% of the area of antral IMAs) deep in the circular muscle (∼6μm above the submucosa). Separate arbors of IMAs in the sphincter interdigitated and overlapped to form a 360° band of mechanoreceptors encircling the pyloric canal. The annulus of vagal IMA arbors, putative stretch receptors tightly intercalated in the sphincter ring and situated near the lumen of the pyloric canal, creates an architecture with the potential to generate gut reflexes on the basis of pyloric sensory maps of high sensitivity and fine spatial resolution.

To supply a fuller morphological characterization of the vagal afferents innervating the lower esophageal sphincter (LES), specifically to label vagal terminals in the tissues forming the LES in the gastroesophageal junction, the present experiment employed injections of dextran biotin into the nodose ganglia of rats. Four types of vagal afferents innervated the LES. Clasp and sling muscle fibers were directly and prominently innervated by intramuscular arrays (IMAs). Individual IMA terminals subtended about 16° of arc of the esophageal circumference, and, collectively, the terminal fields were distributed within the muscle ring to establish a 360° annulus of mechanoreceptors in the sphincter wall. 3D morphometry of the terminals established that, compared to sling muscle IMAs, clasp muscle IMAs had more extensive arbors and larger receptive fields. In addition, at the cardia, local myenteric ganglia between smooth muscle sheets and striated muscle bundles were innervated by intraganglionic laminar endings (IGLEs), in a pattern similar to the innervation of the myenteric plexus throughout the stomach and esophagus. Finally, as previously described, the principle bundle of sling muscle fibers that links LES sphincter tissue to the antropyloric region of the lesser curvature was innervated by exceptionally long IMAs as well as by unique web ending specializations at the distal attachment of the bundle. Overall, the specialized varieties of densely distributed vagal afferents innervating the LES underscore the conclusion that these sensory projections are critically involved in generating LES reflexes and may be promising targets for managing esophageal dysfunctions.

It is well documented that the intrinsic enteric nervous system of the gastrointestinal (GI) tract sustains neuronal losses and reorganizes as it ages. In contrast, age-related remodeling of the extrinsic sympathetic projections to the wall of the gut is poorly characterized. The present experiment, therefore, surveyed the sympathetic projections to the aged small intestine for axonopathies. Furthermore, the experiment evaluated the specific prediction that catecholaminergic inputs undergo hyperplastic changes. Jejunal tissue was collected from 3-, 8-, 16-, and 24-month-old male Fischer 344 rats, prepared as whole mounts consisting of the muscularis, and processed immunohistochemically for tyrosine hydroxylase, the enzymatic marker for norepinephrine, and either the protein CD163 or the protein MHCII, both phenotypical markers for macrophages. Four distinctive sympathetic axonopathy profiles occurred in the small intestine of the aged rat: (1) swollen and dystrophic terminals, (2) tangled axons, (3) discrete hyperinnervated loci in the smooth muscle wall, including at the bases of Peyer's patches, and (4) ectopic hyperplastic or hyperinnervating axons in the serosa/subserosal layers. In many cases, the axonopathies occurred at localized and limited foci, involving only a few axon terminals, in a pattern consistent with incidences of focal ischemic, vascular, or traumatic insult. The present observations underscore the complexity of the processes of aging on the neural circuitry of the gut, with age-related GI functional impairments likely reflecting a constellation of adjustments that range from selective neuronal losses, through accumulation of cellular debris, to hyperplasias and hyperinnervation of sympathetic inputs.

Little is known about the architecture of the vagal motor units that control esophageal striated muscle, in spite of the fact that these units are necessary, and responsible, for peristalsis. The present experiment was designed to characterize the motor neuron projection fields and terminal arbors forming esophageal motor units. Nucleus ambiguus compact formation neurons of the rat were labeled by bilateral intracranial injections of the anterograde tracer dextran biotin. After tracer transport, thoracic and abdominal esophagi were removed and prepared as whole mounts of muscle wall without mucosa or submucosa. Labeled terminal arbors of individual vagal motor neurons (n = 78) in the esophageal wall were inventoried, digitized and analyzed morphometrically. The size of individual vagal motor units innervating striated muscle, throughout thoracic and abdominal esophagus, averaged 52 endplates per motor neuron, a value indicative of fine motor control. A majority (77%) of the motor terminal arbors also issued one or more collateral branches that contacted neurons, including nitric oxide synthase-positive neurons, of local myenteric ganglia. Individual motor neuron terminal arbors co-innervated, or supplied endplates in tandem to, both longitudinal and circular muscle fibers in roughly similar proportions (i.e., two endplates to longitudinal for every three endplates to circular fibers). Both the observation that vagal motor unit collaterals project to myenteric ganglia and the fact that individual motor units co-innervate longitudinal and circular muscle layers are consistent with the hypothesis that elements contributing to peristaltic programming inhere, or are "hardwired," in the peripheral architecture of esophageal motor units.